Preventing desensitization of receptors

ABSTRACT

Desensitization of receptors that control disease is prevented by inhibiting G-protein receptor kinases. This has applicability, e.g., for patients with heart failure or on a left ventricular heart device or a heart pump after surgery or about to undergo surgery and at high risk for a cardiac event or on an opiate or addicted to opiate or with cystic fibrosis or rheumatoid arthritis.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This is a continuation-in-part of U.S. application Ser. No.09/986,807 filed on Nov. 13, 2001, the whole of which is incorporatedherein by reference.

TECHNICAL FIELD

[0002] The invention herein is directed to prevention of desensitizationof receptors to activation by agonists.

BACKGROUND OF THE INVENTION

[0003] Many receptors which are involved in controlling pathologicconditions are coupled to G-proteins (Pierce, K. L., et al, NatureReview (Molecular Cell Biology) 3, 639-650 (2002)). These are calledG-protein coupled receptors (GPCRs). The GPCRs include α-adrenergicreceptors, β-adrenergic receptors, opioid receptors and prostaglandinreceptors. Over time, when agonists are administered to activate thereceptors, the receptors become desensitized, i.e., agonistadministration no longer results in therapeutic activation of receptorsand the receptors regardless of agonist administration are unable tocontrol the pathologic condition.

SUMMARY OF THE INVENTION

[0004] It is known that when agonist binds to a GPCR to activate it, thesequence of events is that the receptor is phosphorylated, thephosphorylated receptor moves to the interior of the cell it isassociated with, i.e., it is internalized, with the internalizationoften involving recruitment of β-arrestin and then the receptor isrecyled and moves to the surface of the cell housing it where it isavailable to control a disease event and to bind to agonist foractivation for control of the disease event. The GPCRs have G-proteinreceptor kinases (GRKs) associated with them. The GRKs phosphorylateagonist-occupied receptors thereby promoting binding of β-arrestinmolecules which inhibit interactions between the receptors andG-proteins while also promoting internalization of the receptors. GRKsthus dampen signaling by the GPCRs. The typical response is decreasedlevel of GPCRs and desensitization thereof, (i.e., inability of agonistto activate the receptor and inability of the GPCRs to control thedisease event). It has been discovered herein that nitric oxide donors(NO donors) that donate nitric oxide or a related redox species andprovide bioactivity that is identified with nitric oxide, preferablyS-nitrosoglutathione (GSNO), inhibit the GRKs dramatically therebyallowing GPCRs to signal and to be recycled to the cell surface, i.e.,thus preventing desensitization of the GPCRs and allowing GPCRs to beavailable in sufficient amount to control the disease event. It has alsobeen discovered herein that administration of GSNO results in growth ofheart muscle (hypertrophy) in vivo (which can be both dependent andindependent of expression of receptors) and prevents cardiacβ-adrenergic receptor down regulation after chronic administration of aβ-adrenergic agonist.

[0005] The above discoveries support the invention herein which isdirected to a method for treating a patient with a disease or pathologiccondition associated with G-protein receptor kinase activity where theG-protein receptor kinase activity would otherwise cause desensitizationof a receptor controlling said disease or condition, said methodcomprising the step of administering NO donor that donates nitric oxideor a related redox species and provides bioactivity that is identifiedwith nitric oxide to inhibit the G-protein receptor kinase activity,thereby sensitizing or preventing desensitization of said receptor.

[0006] The term “disease or condition associated with G-protein receptorkinase activity” is used herein to mean a disease or condition resultingfrom under-stimulation of a GPCR or related insufficient activation of aGPCR.

[0007] G-protein receptor kinase activity is described in Pierce, et al,cited above.

[0008] The term “controlling said disease or condition” is used hereinto mean influence the biochemical or clinical correlate of the diseaseor condition.

[0009] The term “desensitization of a receptor” is used herein to meandecreased activity or decreased level of expression or decreasedresponsiveness.

[0010] The term “to inhibit the G-protein receptor kinase activity” isused herein to mean to decrease its activity.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011]FIG. 1 is a graph of heart weight divided by body weight versuscontrol or agent introduced and shows results of Background Example 2.

[0012]FIG. 2 is a graph of airway resistance versus allergen challengeat recited concentrations and shows results of Background Example 3.

[0013]FIG. 3 is a graph showing effect of GSNO on GRK phosphorylation ofa synthetic peptide and shows results of Background Example 4.

DETAILED DESCRIPTION

[0014] We turn now to the method of the invention herein which isdirected to a method for treating a patient with a disease or pathologiccondition associated with G-protein receptor kinase activity where theG-protein receptor kinase activity would otherwise cause desensitizationof a receptor controlling said disease or condition, said methodcomprising the step of administering NO donor that donates nitric oxideor a related redox species and provide bioactivity that is identifiedwith nitric oxide, to inhibit G-protein receptor kinase activity therebysensitizing or preventing desensitization of said receptor.

[0015] The receptors controlling the diseases or conditions arethiol-containing receptors and can be, for example, β-adrenergicreceptors, α-adrenergic receptors, μ-opioid receptors and prostaglandinreceptors.

[0016] Diseases or conditions where a receptor controlling the diseaseor condition is a β-adrenergic receptor include, for example, right orleft heart failure, a patient on a left ventricular heart assist deviceawaiting heart transplant and a patient who has had heart surgery andotherwise cannot be disconnected from a heart pump without loss of heartfunction or a patient undergoing surgery who is at risk for a cardiacevent. Excluded from invention herein are treatment of pulmonaryhypertension and treatment of systemic hypertension which are covered inthe parent application hereto.

[0017] Diseases or conditions where a receptor controlling the diseaseor condition is an α-adrenergic receptor include benign prostatichypertrophy and urinary incontinence.

[0018] Patients with diseases or conditions where a receptor controllingthe disease or condition is the μ-opioid receptor include patients beingtreated with an opiate because of pain or a patient addicted to anopiate. Patients being treated with opiates include patients with severepain because of surgery, cancer or accidental injury. Opiates include,for example, morphine, oxycodone, codeine and meperidine.

[0019] Diseases or conditions wherein a receptor controlling the diseaseor condition is the prostaglandin receptor include inflammatoryconditions including rheumatoid arthritis. Cystic fibrosis is includedin the case when the NO donor is one capable of acutely lowering FEV1 bymore than 12% and the NO donor is administered in a therapeuticallyeffective amount which is insufficient to acutely lower FEV1 by morethan 12% (i.e., use of a dose which does not cause bronchodilation);treatment of a patient with cystic fibrosis with nitrolylating agentwithout regard to limiting dosage, is taught in WO 02/32418.

[0020] As indicated above the agents administered herein to inhibit GRKactively are NO donors. An NO donor donates nitric oxide or a relatedredox species and more generally provides nitric oxide bioactivity, thatis activity which is identified with nitric oxide, e.g., vasorelaxationor stimulation or inhibition of a receptor protein, e.g., ras protein,adrenergic receptor, NFκB. NO donors including S-nitroso, O-nitroso,C-nitroso and N-nitroso compounds and nitro derivatives thereof andmetal NO complexes, but not excluding other NO bioactivity generatingcompounds, useful herein are described in “Methods in Nitric OxideResearch,” edited by Feelisch, M., and Stamler, J. S., John Wiley &Sons, New York, 1996, pages 71-115 which is incorporated herein byreference. NO donors which are C-nitroso compounds where nitroso isattached to a tertiary carbon which are useful herein include thosedescribed in U.S. Pat. No. 6,359,182 and in WO 02/34705. Examples ofS-nitroso compounds including S-nitrosothiols useful herein include, forexample, S-nitrosoglutathione (GSNO), S-nitroso-N-acetylpenicillamine,S-nitroso-cysteine and ethyl ester thereof, S-nitroso cysteinyl glycine,S-nitrosogamma-methyl-L-homocysteine, S-nitroso-L-homocysteine,S-nitroso-gamma-thio-L-leucine, S-nitroso-delta-thio-L-leucine, andS-nitrosoalbumin. Examples of other NO donors useful herein are sodiumnitroprusside (nipride), ethyl nitrite, nitroglycerin, SIN1 which ismolsidomine, furoxamines, N-hydroxy (N-nitrosamine) and perfluorocarbonsthat have been saturated with NO or a hydrophobic NO donor. A preferredNO donor for use herein is GSNO. When GSNO is administered in thetreatment of a disease or pathologic condition where the receptorcontrolling said disease or condition is a β-adrenergic receptor, theGSNO functions to sensitize or prevent desensitization of the receptoras well as to increase heart pumping action as well as to provide theeffect of an α-adrenergic agonist of maintaining or increasing bloodpressure and also to activate ryanodine receptor so it releases calciumresulting in improved contractility.

[0021] The amount of NO donor administered is an amount which causes thereceptor to control the disease or pathologic condition controlled by itand sensitizes or prevents desensitization of said receptor. An amountthat sensitizes or prevents desensitization of a receptor can bedetermined by pharmacological or clinical response or by receptorbinding studies. The dosage of NO donor administered in the case of apatient with heart failure, or who is connected to a left ventricularheart assist device awaiting heart transplant or who has had heartsurgery and otherwise cannot be disconnected from a heart pump withoutloss of heart function, is a dosage that increases heart pumpingfunction and in the case of GSNO is a dosage which also stimulatesgrowth of heart muscle. The dosage of NO donor for an operativeprocedure is one which protects from a coronary event. The dosage of NOdonor for a patient being treated with or who is addicted to an opiateis a dosage which stabilizes or reduces the dosage of opiate to obtain aparticular level of effect. The dosage of NO donor for an inflammatorycondition is an inflammation ameliorating effect, e.g., to amelioratebronchoconstriction and hypoxia and prevent the long term deteriorationof lung function, dyspnea, cough, chronic airway infection,bronchiectasis, atelectasis and pneumonothorax in patients with cysticfibrosis and in the case of rhematoid arthritis to reduce jointinflammation and tenderness and synovial thickening and joint stiffness.

[0022] In general, administering an effective amount of NO donorinvolves administration to achieve an amount of NO donor in the blood of100 picomolar to 100 micromolar depending on the agent administered andthe disease or condition treated.

[0023] Preferably the NO donor is administered in a therapeuticallyeffective amount which is insufficient to lower mean arterial bloodpressure or pulmonary artery pressure by more than 10%.

[0024] When GSNO is the treating agent, it is preferably administered ata dosage ranging from 0.01 nmol/kg/min to 1000 nmol/kg/min, verypreferably from 0.1 to 10 nmol/kg/min. For diseases or conditions wherea receptor controlling the disease or condition is a β-adrenergicreceptor, the GSNO or NO donor is preferably administered in amountwhich both increases the pumping action of the heart and causes growthof heart muscle and very preferably in conjunction with a βadrenergicreceptor agonist, e.g., isoproterenol, dobutamine or dopamine. Theβadrenergic receptor agonist is preferably administered in an amountwhich increases pumping action of the heart. For isoproterenol, thisdosage ranges from 0.5 to 50 μg/min.

[0025] Administration of the NO donor treating agents have benefit inthe case of patient with chronic heart failure regardless of whetherβ-adrenergic agonist has or is being administered. It is known that inchronic heart failure, catecholamines are highly elevated and thus thereceptor may be down regulated and/or the system desensitized, and thatβ-adrenergic agonists and other inotropic agents can kill patients, ifused chronically because of further down regulation and desensitization.Thus NO donor functioning as a GRK inhibitor, used without β-adrenergicagonist administration, provides a unique therapeutic benefit. Moreover,as indicated above, NO donor treating agent has a benefit in the case ofpatients with chronic heart failure, when administered conjointly withβ-adrenergic agonist.

[0026] The invention is supported by the following background examplesand illustrated by the following working examples:

BACKGROUND EXAMPLE 1

[0027] Mice were treated with 10 ng/kg/day of GSNO or PBS infusedcontinuously for 1 week through an azlet miniosmotic pump placedsubcutaneously. Another set of mice was treated for 7 days withisoproterenol (30 mg/kg/day), and the fourth and final group receivedfor 7 days, isoproterenol (30 mg/kg/day) plus GSNO (10 ng/kg/day). Themice were sacrificed and heart tissue was removed. Saturation bindingwith radiolabeled I¹²⁵-cyanopindolol was carried out in purified cardiacmembrane according to the procedure of Iaccarino et al, Circulation 98,17A3-17A9 (1998). For mice not treated with GSNO or isoproterenol, theamount of β-adrenergic receptor was determined to be 40-50 fmol per mgprotein. For mice not treated with GSNO but treated with isoproterenol(30 mg/kg/day), the amount of β-adrenergic receptor was determined to be20 fmol per milligram protein, demonstrating receptor down-regulation.For mice treated with GSNO but not isoproterenol, the amount ofβ-adrenergic receptor was determined to be 40 fmol per mg/protein or thesame as for normal mice (no GSNO or isoproterenol). For mice treatedwith GSNO and isoproterenol (30 mg/kg/day), the amount of β-adrenergicreceptor was determined to be 90 fmol per mg protein. The experimentshows that GSNO prevents desensitization and down-regulation ofβ-adrenergic receptors caused by isoproterenol and stimulates expressionof the β-adrenergic receptors.

BACKGROUND EXAMPLE 2

[0028] Mice were treated with 10/ng/kg/day of GSNO, or with PBS(control) or with isoproterenol (at a dosage of 30 mg/kg/day) or with 10mg/kg/day of GSNO plus 30 mg/kg/day isoproterenol, infused continuouslyfor 1 week. The animals were sacrificed and the hearts were weighed anda ratio of heart weight to body weight was determined for each case. Theresults are set forth in FIG. 1 where hw:bw, i.e., the legend on thevertical axis, is the ratio of heart weight to total body weight and“ISO” means isoproterenol. As shown in FIG. 1, GSNO treatment increasesthe size of the heart muscle consistent with preventing desensitizationof the β-adrenergic receptor. Treatment with isoproterenol alsoincreased the size of the heart muscle. The combination of isoproterenolplus GSNO has even more effect in increasing size of heart muscle. Theincrease in size of heart (hypertrophy) is an advantage especially inthe case of a patient on a left ventricular heart assist device awaitingheart transplant and in the case of a patient who has had heart surgeryand cannot be disconnected from a heart pump without loss of heartfunction. The asterisks in FIG. 1 mean p>0.05 compared to control.

BACKGROUND EXAMPLE 3

[0029] Mice having normal GSNO reductase (wild type or WT) and GSNOreductase knockout mice (KO), that is with GSNO reductase knocked outthereby having increased level of GSNO, were tested for responsivenessto airway provocation. The mice were challenged with phosphate bufferedsaline (PBS) or ovalbumin (OVA) according to the procedure of describedin Drazen, J., et al, Annual Rev. Physiology 61, 593-625 (1999). Micewere sensitized to ovalbumin (OVA) by intraperitoneal injection 2 weeksprior to airway challenge. Baseline airway resistance measurements(PenH) were performed on mice after methacholine challenge (MCh)followed by airway challenge 24 hours later with aerosolized PBS or OVA.The mice challenged with aerosolized saline (PBS) served as the controlgroup. The results are shown in FIG. 2 where PenH is airway resistanceand MCh is methacholine challenge. As shown in FIG. 2, the wild typemice showed a significant increase in airway resistance after OVAchallenge at methacholine concentrations of 12 and 25 mg/ml whereas theKO mice had no change. The results suggest that GSNO administration willcause hyporesponsiveness to allergen challenge further suggesting thatthe GSNO causes sensitizing or prevents desensitization of theappropriate GPCRs, e.g., prostaglandin receptors.

BACKGROUND EXAMPLE 4

[0030] An isolated system was provided consisting of medium, GRK,standard synthetic peptide substrate and radiolabeled ³²P-adenosinetriphosphate, i.e., ³²P-ATP, as a source of phosphate. Runs were carriedout on the system without additives, and with 500 μM glutathione (GSH)present and with GSNO (500 μM) present or with GSNO (50 μM) present orwith GSNO (5 μm) present. The amount of bound ³²P was measured in countsper minute (CPM). The results are shown in FIG. 3 where there is shown aconcentration dependent reduction in incorporation of ³²P when GSNO ispresent. The results suggest that GSNO inhibits GRK to inhibitphosphorylation of substrate (incorporated ³²P). The same result of GRKinhibition and interference with substrate phosphorylation was foundwhen S-nitroso-cysteine (L-SNC) was substituted for GSNO.

EXAMPLE I

[0031] A 65-year old with class 4 congestive heart failure enters thehospital with shortness of breath and bradyacardia. He is given 10 μgper minute of intravenous isoproterenol with resolution of symptoms. Thepatient is subsequently switched to IV dobutamine at 10 μg per kg perminute titrated to cardiac output. Over the following three days thedrug is increased to 30 μg per kg per minute to maintain cardiac output.The patient is then begun on IV GSNO at 2 nmol per kg per minute andcardiac output improved over the following 24 hours. The dose ofdobutamine is subsequently decreased gradually and then stopped. Thepatient undergoes bypass surgery and does well even though he has beenviewed as at high risk.

EXAMPLE II

[0032] A 49-year old white male awaiting cardiac transplantation isplaced a left ventricular assist device, but continues to suffer fromsevere shortness of breath. He is begun on IV GSNO 4 nmol per kg perminute with decrease in shortness of breath and an increase in cardiacoutput.

EXAMPLE III

[0033] A 68-year old white female with an ejection fraction of 17%undergoes coronary artery bypass grafting. The surgeons are unable toremove the patient from bypass. Intravenous GSNO is begun at 2 nmol perkg per minute and after two hours the patient is successfully removedfrom bypass.

EXAMPLE IV

[0034] A 75-year-old white male with metastatic prostate cancer issuffering from severe bone pain unresponsive to opiates. He is begun onintravenous GSNO at 10 nmol per kg per minute in conjunction withmorphine which now relieves his pain.

EXAMPLE V

[0035] A 29-year-old heroine addict enters a clinic where his dose istapered gradually. In order to avoid symptoms of withdrawal, ethylnitrite is started intravenously at 2 nmol per kg per minute andsymptoms of withdrawal, including sweats and shaking, are ameliorated.

EXAMPLE VI

[0036] A 17-year-old white female with cystic fibrosis enters thehospital complaining of shortness of breath. She is begun on inhaledbeta agonists, but shows little improvement over the following six days.She is started on inhaled GSNO (3 cc of a 10 mM solution, pH 7, 4×/day).Although her FEV1 does not change acutely and her blood pressure remainsstable, symptoms of shortness of breath resolve over the following 48hours (with corresponding improvement in FEV1).

EXAMPLE VII

[0037] A 66-year-old white female with rheumatoid arthritis andpulmonary infiltrates complains of shortness of breath and knee pain.She is begun on intravenous ethyl nitrite at 2 nmol per kg per minutewith improvement in respiratory status and a decrease in knee pain.

[0038] Variations

[0039] Variations on the above will be obvious to those skilled in theart. Therefore the scope of the invention is to be determined by theclaims.

What is claimed is:
 1. A method of treating a patient with a disease orpathologic condition associated with G-protein receptor kinase activitywhere the G-protein receptor kinase activity would otherwise causedesensitization of a receptor controlling said disease or condition,said method comprising the step of administering nitric oxide donor thatdonates nitric oxide or a related redox species and provides bioactivitythat is identified with nitric oxide to inhibit the G-protein receptorkinase activity thereby sensitizing or preventing desensitization ofsaid receptor.
 2. The method of claim 1 where said NO donor isS-nitrosoglutathione.
 3. The method of claim 1 where the receptor is aβ-adrenergic receptor.
 4. The method of claim 3 where the disease orcondition is heart failure.
 5. The method of claim 4 where the NO donoris S-nitrosoglutathione and the S-nitrosoglutathione administrationcauses increase in heart pumping and also stimulates growth of heartmuscle.
 6. The method of claim 5 where the S-nitrosoglutathione isadministered in conjunction with administration of a therapeuticallyeffective amount of isoproterenol.
 7. The method of claim 3 where thedisease or condition is where the patient is connected to a heart assistdevice awaiting heart transplant.
 8. The method of claim 7 where the NOdonor is S-nitrosoglutathione.
 9. The method of claim 3 where thedisease or condition is where the patient has had heart surgery andotherwise cannot be disconnected from heart pump without loss of heartfunction.
 10. The method of claim 9 where the NO donor isS-nitrosoglutathione.
 11. The method of claim 1 where the patient isabout to undergo surgery and is at high risk for a cardiac event. 12.The method of claim 11 where the NO donor is S-nitrosoglutathione. 13.The method of claim 1 where the receptor is the μ-opioid receptor andthe patient is being treated with or is addicted to an opiate.
 14. Themethod of claim 1 where the disease or condition is an inflammatorycondition which is not asthma, and when the inflammatory condition iscystic fibrosis and the NO donor is one capable of acutely lowering FEV1by more than 12%, the NO donor is administered in a therapeuticallyeffective amount which is insufficient to acutely lower FEV1 by morethan 12%.
 15. The method of claim 14 where the disease or condition iscystic fibrosis.
 16. The method of claim 14 where the disease orcondition is rheumatoid arthritis.